Aloe is a simple plant with an elaborate set of biologically active substances. (Cohen et al. in Wound Healing/Biochemical and Clinical Aspects, 1st ed. W. B. Saunders, Philadelphia (1992)). Over 300 species of Aloe are known, most of which are indigenous to Africa. Studies have shown that the biologically active substances are located in three separate sections of the aloe leaf--a clear gel filet located in the center of the leaf, in the leaf rind or cortex of the leaf and in a yellow fluid contained in the pericyclic cells of the vascular bundles, located between the leaf rind and the internal gel filet, referred to as the latex. Historically, Aloe products have been used in dermatological applications for the treatment of burns, sores and other wounds. These uses have stimulated a great deal of research in identifying compounds from Aloe plants that have clinical activity, especially anti-inflammatory activity. (See, e.g., Grindlay and Reynolds (1986) J. of Ethnopharmacology 16:117-151; Hart et al. (1988) J. of Ethnopharmacology 23:61-71). As a result of these studies there have been numerous reports of Aloe compounds having diverse biological activities, including anti-tumor activity, anti-acid activity (Hirata and Suga (1977) Z. Naturforsch 32c:731-734), anti-diabetic activity, tyrosinase inhibiting activity (Yagi et al. (1987) Planta medica 515-517), and antioxidant activity (International Application Serial No. PCT/US95/07404, published Dec. 19, 1996, publication number WO 96/40182). Aloe products are also used extensively in the cosmetic industry to protect skin against ultraviolet light. (Strickland et al. (1994) J. Invest. Dermatol. 102:197; Grollier et al. U.S. Pat. No. 4,656,029, issued Apr. 7, 1987).
The commercial use of Aloe is wide spread, particularly in the cosmetic industry. There are currently many commercially available products which contain Aloe as an ingredient, however, to date there is there is no commercially viable process for the detection or quantitation of the amount of Aloe in these products. In order to better regulate this industry, there is currently a need for a process to detect and quantitate the amount of Aloe present in a mixture. At present manufacturers and distributors of Aloe can adulterate and dilute Aloe products and go undetected. It is imperative to develop standards to protect the Aloe product name.
The Polymerase Chain Reaction (PCR), is a recently developed technique which has had a significant impact in many areas of science. PCR is a rapid and simple method for specifically amplifying a target DNA sequence in an exponential manner. (Saiki et al. (1985) Science 230:1350; Mullis and Faloona (1987) Methods Enzymol. 155:335). Briefly, the method consists of synthesizing a set of primers that have nucleotide sequences complementary to the DNA that flanks the target sequence. The primers are then mixed with a solution of the target DNA, a thermostable DNA polymerase and all four deoxynucleotides (A, T, C and G). The solution is then heated to a temperature sufficient to separate the complementary strands of DNA (approximately 95.degree. C.) and then cooled to a temperature sufficient to allow the primers to bind efficiently and specifically to the flanking sequences. The reaction mixture is then heated again (to approximately 72.degree. C.) to allow the DNA synthesis to proceed. After a short period of time the temperature of the reaction mixture is once again raised to a temperature sufficient to separate the newly formed double-stranded DNA, thus completing the first cycle of PCR. The reaction mixture is then cooled and the cycle is repeated. Thus, PCR consists of repetitive cycles of DNA melting, annealing and synthesis. Twenty replication cycles can yield a million fold amplification of the target DNA sequence. The ability to amplify a single DNA molecule by PCR has applications in environmental and food microbiology (Wernars et al. (1991) Appl. Env. Microbiol. 57:1914-1919; Hill and Keasler (1991) Int. J. Food Microbiol. 12:67-75), clinical microbiology (Wages et al. (1991) J. Med. Virol. 33:58-63; Sacramento et al. (1991) Mol. Cell Probes 5:229-240; Laure et al. (1988) Lancet 2:538), oncology (Kumar and Barbacid (1988) Oncogene 3:647-651; McCormick (1989) Cancer Cells 1:56-61; Crescenzi et al. (1988) Proc. Natl. Acad. Sci. USA 85:4869), genetic disease prognosis (Handyside et al. (1990) Nature 344:768-770), blood banking (Jackson (1990) Transfusion 30:51-57) and forensics (Higuchi et al. (1988) Nature (London) 332:543). PCR has also been used in phylogenetic studies of various organisms. For example, PCR has been used to authenticate and differentiate three medical species of Panax from one another and also from their common adulterants. (Shaw and But (1995) Planta Med. 61:466-469; Cheung et al. (1994) J. Ethnopharmacol. 42:67-69).
The feasibility of using the polymerase chain reaction to identify Aloe depends on establishing specific primers that detect Aloe DNA and extraction protocols that reproducibly recover the DNA. The proportion of Aloe DNA which survives processing and the successful recovery of DNA and elimination of PCR inhibitory substances from DNA extracts will dictate the sensitivity of the PCR reaction necessary to detect Aloe DNA in extracts of industrial Aloe products. The sensitivity of the PCR reaction that is required defines the type of PCR-product detection-system that is necessary and the type of containment required to eliminate spurious results and possible contamination.